Method for Producing Recombinant Stem Bromelain

a technology of stem bromelain and recombinant protein, which is applied in the direction of hydrolases, biochemistry apparatus and processes, enzymes, etc., can solve the problems of high material and operation costs, and achieve the effect of high production rate and cost-effectiveness

Inactive Publication Date: 2011-06-23
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008]It is also an object of the present invention to provide a cost effective method for producing high amounts of stem bromelain.

Problems solved by technology

Unfortunately, all these attempts require high cost of materials and operation.

Method used

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  • Method for Producing Recombinant Stem Bromelain
  • Method for Producing Recombinant Stem Bromelain
  • Method for Producing Recombinant Stem Bromelain

Examples

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Method

[0040]About 200 mg fresh maturing pineapple stems (from Ananas comosus N36) were used for RNA isolation. The pineapple stems were weighed and placed in liquid nitrogen, then ground with mortar and pestle to produce a tissue powder. The tissue powder and liquid nitrogen were then decanted into an RNase-free, liquid-nitrogen-cooled, microcentrifuge tube. Once the liquid nitrogen had evaporated, a lysis buffer containing guanidine thiocyanate was immediately added, and the tissue and buffer mixture was vortexed, producing a lysate. Next, the lysate was transferred to a homogenizer and centrifuged for 2 min to remove insoluble plant material, homogenize the lysate and reduce lysate viscosity. The supernatant was collected and transferred to a new microcentrifuge tube before adding 0.5 volume of ethanol (96-100%) to the cleared lysate to create conditions and mixing thoroughly to promote selective binding of RNA to the spin column membrane used for extracting the RNA.

[0041]The lysa...

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Abstract

The present invention relates to a method for producing recombinant stem bromelain. Total RNA is isolated from pineapple plant (11) and used for reverse transcription polymerase chain reaction to generate multiple copies of stem bromelain gene (12). The gene is then cloned into a cloning vector (13), then transformed to a competent cell to produce an entry clone (14). After identifying and selecting the positively transformed entry clone (15), the stem bromelain gene is sub-cloned into a destination vector (16) and transformed into host cells to produce recombinant cells (17). After identifying and selecting the positively transformed recombinant cells (18), the recombinant cells are induced to express recombinant stem bromelain (19). Subsequently, the recombinant cells are harvested and the recombinant stem bromelain purified (20). The recombinant stem bromelain produced is in an active form and demonstrates activity similar to native stem bromelain.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]This invention relates to a method for producing a recombinant protein, and more particularly to a method for producing recombinant stem bromelain.[0003]2. Description of Related Arts[0004]Commercial bromelain is a crude, aqueous extract from the stems and immature fruits of pineapples. Crude pineapple stem preparation contains two distinct cysteine proteases: stem bromelain and ananain. Three cysteine proteases are present in the pineapple stem: stem bromelain, ananain, and comosain. Bromelain is a protein-digesting enzyme similar in function to papain and ficin. It is also the most cost effective natural protease.[0005]Bromelain is known to have a number of uses. A known pharmaceutical application of bromelain is the enzymic debridement of necrotic tissues from ulcers and burn wounds. Other therapeutic benefits include reversible inhibition of platelet aggregation, management of bronchitis, improved healing of surgica...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/50
CPCC12N9/50
Inventor AMID, AZURAYUSOF, FARIDAHISMAIL, NURUL AZIRA
Owner INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
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